Contact sensitivity (CS) responses are very common allergic disorders of the skin. Elicitation of the late T cell 24 hr phase of CS in immunized mice requires an early antigen-specific initiating phase. This peaks 2 hr after challenge and depends on local complement activation to elaborate C5a due to antigen binding IgM antibodies produced by B- I cells, leading to vascular activation for local T cell recruitment. We hypothesize that NK T cell derived IL-4 activates the B-1 cells to mediate this CS-initiation. Aim #1: Determine the role of IL-4 in the early CS response. CS responses in IL- 4-/- vs. wild type mice will be investigated. If IL-4-/- mice show an impaired early response, the deficient mice will be injected with different doses of IL-4. Activated immune B-1 cells from contact sensitized wild type mice will be transferred into sensitized IL-4 deficient mice and test for elicitation of CS. Mice with defective IL-4 signaling (Stat-6-/-) will also be evaluated similarly. Aim #2: Evaluate contact sensitivity in NK T cell- deficient mice. If IL-4 is found involved in CS-initiation, NK T cell deficient mice will be evaluated. Two strains of NK T cell deficient mice will be employed: CDld-/- and Jalpha281-/-. If there is deficient CS, then adoptive cell transfers of FACS purified NK T cells (using CD1d fluorescent tetramers) and B-1 cells (CD 19+ CD5+) will be performed, as well as treatment with different doses of IL-4. Aim #3: Analysis of the effects of IL-4 on activation of B-1 cells. The phenotype of the B-1 cells will be analyzed for evidence of activation induced by IL-4. The surface marker Thy-1, expressed in vitro on B cells after IL-4 treatment, and on activated CS-initiating cells, will be analyzed on B-1 cells in CS of wild type mice, as well as in CDld-/- and J281-/- mice. CD23 expression, generally associated with IL-4 activation will be investigated similarly. Further the effect of the NK T cell non-specific activator alpha-GalCer, on activation of B-1 cells also will be evaluated. Summary: The current proposal will investigate the role of IL-4 produced by NK T cells in initiating CS responses. We hypothesize that NK T cellderived IL-4 activates B-1 cells to secrete specific IgM, lading to subsequent Ag- dependent elaboration of C5a to initiate CS. Recognized only a few years ago, NK T cells have not been associated with contact dermatitis, nor with the initiating phase of in vivo T cell mediated immunity, nor with allergies. Furthermore, our studies may have the important result of identifying a physiological ligand for the semi-invariant alpha/beta-TCR on regulatory NK T cells.